It is well known, that the in-vitro stability and in-vivo half-life of polypeptides can be increased by covalent attachment of biocompatible polymers (in the following referred to as conjugation or modification). Modification of the polypeptide surface also has the advantage of decreasing the immunogenicity exhibited by the polypeptide.
Pegylation, i.e. coupling of various polyethyleneglycols (PEG) to a polypeptide, is a technique widely used for increasing the in-vitro stability and in-vivo half-life of e.g. proteins. In pegylation, many techniques have been proposed over the years. Reference is here made to Zalipsky, S. et al in Poly(Ethylene Glycol) Chemistry, Biotechnical and Biomedical Applications, Plenum, N.Y. (1992), and Katre N. V., Adv. Drug Deliv. Rev., 10 91-114 (1993).
For some polypeptides, a loss of activity or function has been recognized as a consequence of this conjugation, an effect that increases by degree of modification (Inada, Y. et al, Trends in Biotechnology, 13, 86-91 (1995)). Methods have been developed for making coupling more selective, to circumvent this problem. Site-directed mutagenesis for instance, has been applied for recombinant interleukin-2 (rIL-2). A specific target can be created by insertion of a cystein (Goodson, R. J. and Katre, N. V., Bio/Technology 8, 344-346 (1990); Katre 1993, see above). Such a route is not generally applicable for therapeutic proteins, since amino acid substitution may change the original characteristics of the molecule, and is therefore controversial. Alternatively, the polymer can be directed towards glycosylated sites of a protein, e.g. factor IX as disclosed in WO 94/29370 (Enzon). This involves, however, oxidation of the carbohydrate moieties which can be achieved by reacting the glycoprotein with sodium periodate or enzymatically by galactose oxidase. These conditions are often detrimental for the molecule. In this particular case, the glycosylated sites intended for conjugation were located at a factor IX peptide sequence which is removed during proteolytic activation in-vivo. Hence, the biocompatible polymer does not influence the function of the active polypeptide.
In WO 94/13322 (Farmitalia Carlo Erba) it is shown that pegylation can be carried out without impairing the function of certain sites essential for the function of the particular protein ("first substance"). This is achieved by protecting the sites by contacting the first substance with a second substance which specifically binds to the said sites. More particularly, the pegylation is carried out by immobilizing the particular protein on a resin with ligands having specific affinity to the said protein. Second substances are for instance complementary biological molecules. Examples of couples disclosed in WO 94/13322 are antibody (first substance)--corresponding antigen (second substance); specific inhibitor (first substance)--enzyme (second substance); growth factor (first substance)--corresponding receptor (second substance), or the reverse of each of these couples.
In a process intended for pharmaceutical production it is however advantageous if the use of substances of biological complexity can be kept at a minimum. This is primarily due to the strict requirements for documentation on biochemical homogeneity and safety for the use of said substances. Therefore, the use of affinity ligands produced by organic-chemical synthesis would be advantageous.
DE 3717210 relates to a process for modification of biopolymers, preferably charge-carrying biopolymers, by immobilizing the biopolymer on for instance an ion-exchange adsorbent, reacting the biopolymer with reagents, e.g. enzymes or other biochemical reagents, for obtaining a reaction product and subsequently desorbing the reaction product from the adsorbent. The reaction product is preferably a nucleic acid cleaved with a restriction enzyme. In DE 3717210 the adsorbed state of the biopolymer is utilized to better expose the biopolymer to reagents, thereby increasing the efficiency of the modification. There is no indication of shielding exposed targets nor a purpose of retaining activity of the biopolymer, that would come to advantage for a function of the biopolymer in-vivo. The invention of DE 3717210 is merely aimed at mapping properties of biomolecules, such as nucleic acids, changing the original character by actual processing or increasing the number of functional entities of the macromolecule, such as incorporation of radioactive isotopes.
Many proteins intended for therapeutic use have been conjugated, commonly by use of various pegylation techniques (Francis, G.E. et al, in Stability of Protein Pharmaceuticals/Series: Pharmaceutical Biotechnology 3, 235-263 (1992); Inada, Y. et al, Trends in Biotechnology, 13, 86-91 (1995)). Most examples concern intravenous administration. However, uptake after subcutaneous administration to plasma, lung, liver, and spleen of some mPEG-conjugated allergens has been disclosed and immunotherapy with mPEG-conjugated allergens given subcutaneously has proven to be effective (Dreborg, S. and .ANG.kerblom, E. B., Crit. Rew. Therap. Drug Carr. Sys. 6(4), 315-365 (1990)). Also, intramuscular administration has been used in clinical trials of adenosine deaminase (Hershfield, M. S. et al, New Eng. J. Med. 316, 589-596 (1987). Pegylation has also been claimed, in a few cases, to be beneficial for the oral route. Thus, pegylation lation of IgG for oral administration has been disclosed in EP-A-0 614 373 to Mount Sinai School of Medicine. Pegylation of factor VIII and factor IX for oral administration has been disclosed in Sakuragawa et al, Acta Med. Biol., 34(3), 77-84 (1987) and in Japanese Patent Application No. 44509/83 to Nippon Chemifar Company.
Factor VIII is a protein which participates in the intrinsic blood coagulation. It is a cofactor in the reaction where the enzyme factor IXa in the presence of phospholipid and calcium ions converts the proenzyme factor X to the active form, factor Xa, ultimately leading to a fibrin clot. Human factor VIII is synthesized as a single-chain molecule of approximately 300 kDa and consists of the structural domains A1-A2-B-A3-C1-C2 (Gitschier et al., 1984, Nature 312, p. 326; Wood et al., 1984, Nature 312, p. 330; Vehar et al., 1984, Nature 312, p. 337; Toole et al., 1984, Nature, 312, p.342). The precursor product is processed into two polypeptide chains of 200 and 80 kDa in the Golgi and the two chains held together by metal ion(s) are expressed in the blood (Kaufman et al., 1988, J. Biol. Chem., 263, p. 6352; Andersson et al., 1986, Proc. Natl. Acad. Sci., 83, p. 2979). The B-domain of factor VIII seems to be dispensable as regards the factor VIII cofactor function while the A and C domains have several interaction sites for other macromolecules playing a role in the hemostasis (Sandberg et al., 1993, Thrombos. Haemostas., 69, p. 1204 and Lind et al., 1995, Eur. J. Biochem., 232, p. 19).
The von Willebrand factor (vWf) is a multifunctional polymeric plasma protein consisting of disulfide-linked subunits of about 240 kDa. The subunits form a heterogeneous population of multimers that have a range of molecular weights from c. 1 MDa to 20 MDa. The function of vWf in primary hemostasis is to promote platelet adhesion to the vessel wall in conditions of high shear rate. vWf also binds factor VIII tightly but noncovalently. In normal human plasma factor VIII and vWf circulate complexed to each other. vWf has an important stabilizing effect on the factor VIII molecule in that it protects the molecule from degradation by proteases (Koedam et al., 1987, Doctoral Thesis, ICG Printing, Dordrecht, 1986, p 83; Hamer et al., 1986, Haemostasis, 1987, 58, p. 223). The human in-vivo half-life of factor VIII is usually 10-15 hours. In vWf deficient patients the reduced levels of vWf are accompanied by reduced levels of factor VIII due to an impaired release and increased rate of degradation of factor VIII. Tuddenham et al., 1982, Br. J. Haematol., 52, p. 259 and Brinkhous et al., 1985, Proc. Natl. Acad. Sci., 82, p. 8752 showed that the presence of vWf has an important effect on the in-vivo survival of factor VIII. When factor VIII was infused in hemophilic dogs a half-life of 7-10 hours was obtained while the half-life was c. 1 hour after infusion in vWf deficient dogs (Brinkhous et al., see above).
Factor IX is the proenzyme of factor IXa described above. It is a serine protease and is one of the vitamin K dependent coagulation proteins. The molecular mass is about 55 kDa (DiScipio et al., 1977, Biochemistry, 16, p. 698). Factor IXa interacts specifically with other components participating in the factor X activation (In: Haemostasis and Thrombosis, 1994, vol. 1, third edition, ed. Bloom A. et al.)
It is evident from the above paragraphs, that factor VIII is a protein with several interaction sites, each responsible for a specific function. Therefore, it is difficult to modify factor VIII with fully retained biological function.
The pegylation technique has been applied previously to protein mixtures containing factor VIII. Thus, it has been disclosed in WO 94/15625 (Enzon) that pegylation of factor VIII using a carbamate (urethane) linkage at an undetermined modification degree resulting from a 100 fold molar excess of mPEG relative to factor VIII, can increase the in-vivo half-life in mice from 13 hours to 55 hours. Conventionally, the half-life in mice is considered to be about 1 hour. Thus, 13 hours is an unusually long half-life in mice. Furthermore, it must be taken into account, that with the extremely low purity of the starting factor VIII preparation (20-50 IU/mg protein), other proteins than factor VIII were predominant during the coupling reaction. One important protein usually present in factor VIII preparations of low purity is the von Willebrand factor, which has a stabilizing function for factor VIII and could also very well contribute to the protection of the corresponding functional site during the process of conjugation. After conjugation, mPEG can be located on any of the proteins present in the protein mixture of which factor VIII normally constitutes only a small portion. The pegylated protein mixture containing factor VIII was used for intravenous administration. A well defined starting material, however, is one of the essential factors constituting the controlled conditions required for pharmaceutical production.
Other polymers have also been used for conjugating factor VIII. Thus, it has been disclosed in U.S. Pat. No. 4,970,300, that dextran conjugation can be applied to prolong the half-life of factor VIII.
In most coupling techniques, the polymer reagent reacts with .epsilon.-amino groups of lysine residues of the polypeptide. These are often spread all over the polypeptide surface, and may very well result in a conjugation adjacent to a functional site. As a consequence, by random coupling, the activity or function is often disturbed. It is our experience, that when applying such techniques to very pure preparations, such as a B domain-deleted recombinant factor VIII with coagulant activity, this activity is severely diminished already at a modification degree of about 5 mPEG/factor VIII molecule.